The mechanism of blood coagulation normally occurs in a cascade of two possible routes. One of the routes, the so-called extrinsic blood coagulation, starts with the liberation of thromboplastin and the activation of factor VII. Activated factor VII in turn activates factor X, followed by an activation of factor V and factor II (prothrombin). Factor IIa (thrombin) converts fibrinogen into fibrin at the end of the cascade.
The other route, the so-called intrinsic blood coagulation, occurs via an activation of factor XII by contact with and subsequent activation of factor XI, factor IX and factor X in the presence of calcium and factor VIII, followed by an activation of factor II to factor IIa which triggers the coagulation by cleaving fibrinogen to fibrin. Thus, factor IIa plays a central role in both routes of the blood coagulation cascade. Hitherto, there has been an intensive search for anticoagulants which may particularly be utilized in the treatment of septic shock, thromboses, embolisms, arteriosclerosis and cardiac infarctions, furthermore in case of blood transfusions or following surgery. One method of suppressing the coagulation of blood is the direct administration of substances which inhibit thrombin.
Hitherto, heparin or coumarin have been utilized as anticoagulants. They are, however, relatively systemic and increase the risk of inner hemorrhages. Hirudin, on the other hand, is extremely specific in its binding to thrombin and offers further advantages as compared to the other anticoagulants. It does not require endogenous cofactors, is pharmacodynamically inert, exhibits no effect on blood cells, plasma proteins (with the exception of thrombin) or enzymes, and is immunogenic on account of its small molecular size. Furthermore, hirudin is not stored in organs and is excreted unchanged in urine.
Hirudin is a single-chain polypeptide of 65 amino acids which is naturally formed by the medicinal leech (Hirudo medicinalis) in its secretory glands. Hirudin acts as extremely strongly binding and highly specific inhibitor for the protease thrombin and prevents blood coagulation. The mechanism of the effect of hirudin as thrombin inhibitor has been cleared up: The C-terminal part of hirudin binds to the anion binding sites of the thrombin and thus occupies the binding site of the fibrinogen chain on thrombin. In addition, the N-terminal part of hirudin blocks the active site of thrombin (Szyperski et al. 1992, J. Mol. Biol. 228: 1206-1211; Fenton et al. 1991, Blood Coagul. Fibrinol.2: 69-75; Rydel et al. 1990, Science 249: 277-280; Karshikov et al. 1992, Prot. Science 1: 727-735; Markwardt 1991, Thromb. Haemost. 66: 141-152). For this reason, there has already been an interest for quite some time in using hirudin as a specific anticoagulant.
Recently it has been possible to prepare large amounts of hirudin by a recombinant route, and to use them for pharmacological investigations (Rigel et al. 1993, Circl. Res. 72: 1091-1102; Loison et al. 1988, Biotechnol. 6: 72-77; Zawilska et al. 1993, Thromb. Res. 69: 315-320; Klocking et al. 1990, Blut 60: 129; Fareed and Walenga 1989, FASEB J. 3: 328; Markwardt et al. 1988, Pharmazie 43: 202-207). There result several clinical applications for hirudin: in hemodialysis, as an anticoagulant during the pulmonary transluminal coronary angioplasty (PTCA), for the prophylaxis of post-operative thrombosis, for the prevention of rethrombosis, for microvascular surgery, as anti-coagulant in hemodialysis and in case of extracorporeal circulation, as an admixture to thrombolytic agents, such as, e.g., plasminogen activators and streptokinase, as anticoagulant during surgery and for the clinical suppression of coagulation.
When administering anticoagulants, exact dosing, however, is difficult. For instance, the inhibition of thrombin in the circulation of blood caused by hirudin can lead to undesired complications and hemorrhages requiring an immediate elimination of hirudin from circulation (Fareed et al. 1991, Sem. Thromb. Hemost. 17: 137-144; Bruggener et al. 1989, Pharmazie 44: 648-649; Fareed and Walenga 1989, FASEB J. 3: 328). Yet the determination of the hirudin level (differentiation of free and bound hirudin) in the blood and monitoring the course of the hirudin excretion are possible only indirectly via the determination of the thrombin activity. At present, it is only possible to reduce the hirudin level in blood by natural excretion and, optionally, by means of dialysis. The administration of prothrombin has also been suggested (Walenga et al. Sem. Thromb. Hemost. 15:316:1989), yet the conversion of prothrombin into thrombin is time-dependent in circulation. On the other hand, an excess of thrombin favours the coagulation tendency. Not least of all, hirudin does form a very strong complex with thrombin which is difficult to dissociate even in vitro so that dosing of the hirudin level via a displacement mechanism realistically has not been practicable so far.
Thus there has been an intensive search in the prior art for a suitable antagonist to hirudin which can be used purposefully and thus does not exhibit side effects as regards blood coagulation. Although this has been a known problem of hirudin research (Markwardt F., Haemostasis 21:11; 1991), to date there have not been any practicable solutions which could be used in medicine.
It has been suggested (Bruggener et al., Pharmazie 44:648; 1989) to carry out a chemical change of the thrombin. For this, diisopropyl fluorophosphate that has been purified from plasma was coupled to thrombin. DIP accumulates at the active site of thrombin, thereby changing the three-dimensional structure of the catalytic region. The DIP-thrombin formed is enzymatically inactive, yet binds hirudin. However, diisopropyl fluorophosphate is extremely toxic and dangerous. Since the binding of DIP to thrombin is not very stable, DIP can easily dissociate therefrom. A DIP-thrombin complex disintegrating in vivo thus is completely unsuitable for a clinical application.
In WO 93/15757 prothrombin intermediates have been suggested as antidotes to hirudin. However, these products comprise the usual risks generally inherent in preparations obtained from plasma, e.g. contamination by human pathogenic viruses.
Beside the use of heparin, coumarin and hirudin for preventing blood coagulation, also synthetic thrombin inhibitors, such as NAPAP (Na-(2-naphthyl-sulfurylglycyl)-D,L-amidinophenyl-alanin peptide) or PPACK (D-Phe-Pro-Arg-CHCl) are known. Furthermore, it has i.a. been contemplated to use modified proteins, such as, e.g., inactivated coagulation factors, directly as anticoagulants. There, one particular problem is that in vivo the modified protein possibly could be eliminated from blood more rapidly than the wild type protein. The coagulation process, comprising the cooperation of the intrinsic and extrinsic blood coagulation cascade and cell surface receptors, is very complex. Thus, apart from its greatly reduced or completely inhibited coagulation activity, an inactivated coagulation factor usable in vivo for therapy or prophylaxis should not differ from the natural protein in any further essential property, such as, e.g., receptor binding capacity. An in vivo half-life of the inactive protein corresponding to that of the active coagulation factor or even longer than that would be desirable. Since particularly thrombin has a very short half-life in vivo, an inactive coagulation factor having an extended half-life would increasingly displace the active protein, e.g. thrombin, from its receptor in case of a competitive inhibition. This would have the advantage that merely a relatively low dose would have to be administered for an efficient anticoagulant action of the inactive protein.